Many biological substances, especially nucleic acids, present special challenges in terms of isolating them from their natural environment. On the one hand, they are often present in very small concentrations and, on the other hand, they are often found in the presence of many other solid and dissolved substances e.g. after lysis of cells. This makes them difficult to isolate or to measure, in particular in biospecific assays which allow the detection of specific nucleic acids, or the detection of specific properties of a nucleic acid. Such biospecific assays play a major role in the field of diagnostics and bioanalytics in research and development. Examples for biospecific assays are hybridisation assays, immuno assays and receptor-ligand assays. Hybridisation assays use the specific base-pairing for the molecular detection of nucleic acid analytes e.g. RNA and DNA. Hence, oligonucleotide probes with a length of 18 to 20 nucleotides may enable the specific recognition of a selected complementary sequence e.g. in the human genome. Another assay which entails the selective binding of two oligonucleotide primers is the polymerase chain reaction (PCR) described in U.S. Pat. No. 4,683,195. This method allows the selective amplification of a specific nucleic acid region to detectable levels by a thermostable polymerase in the presence of desoxynucleotide triphosphates in several cycles.
As described above, before the nucleic acids may be analyzed in one of the above-mentioned assays or used for other processes, they have to be isolated or purified from biological samples containing complex mixtures of different components as e.g. proteinaceous and non-proteinaceous components. Often, for the first steps, processes are used which allow the enrichment of the component of interest, i.e. the nucleic acids. Frequently, these are contained in a bacterial cell, a fungal cell, a viral particle, or the cell of a more complex organism, such as a human blood cell or a plant cell. Nucleic acids as a component of interest can also be called a “target component”.
To release the contents of said cells or particles, they may be treated with enzymes or with chemicals to dissolve, degrade or denature the cellular walls and cellular membranes of such organisms. This process is commonly referred to as lysis. The resulting solution containing such lysed material is referred to as lysate. A problem often encountered during the lysis is that other enzymes degrading the target component, e.g. desoxyribonucleases or ribonucleases degrading nucleic acids, come into contact with the target component during lysis. These degrading enzymes may also be present outside the cells or may have been spatially separated in different cellular compartiments before the lysis and come now into contact with the target component. Other components released during this process may be e.g. endotoxins belonging to the family of lipopolysaccharides which are toxic to cells and can cause problems for products intended to be used in human or animal therapy.
In the next steps of the sample preparation which follow on the lysis step, the nucleic acids are further enriched. Nucleic acids are normally extracted from the complex lysis mixtures before they are used in a probe-based assay. There are several methods for the extraction of nucleic acids. Sequence-dependent or biospecific methods include, e.g., affinity chromatography or hybridisation to immobilised probes. Sequence-independent or physico-chemical methods include, e.g., liquid-liquid extraction with phenol-chloroform, precipitation with pure ethanol or isopropanol, extraction with filter paper, extraction with micelle-forming agents as cetyl-trimethyl-ammonium-bromide, binding to immobilized, intercalating dyes such as acridine derivatives, adsorption to substrates such as silica gel or diatomic earths, adsorption to magnetically attractable glass particles (MGP) or organo silane particles under chaotropic conditions. Direct binding of the nucleic acids to a substrate such as a material with a silica surface is preferred because among other reasons the nucleic acids do not have to be modified and even native nucleic acids can be bound.
Particularly interesting for extraction purposes is the adsorption of nucleic acids to a glass surface although other surfaces are possible.
Nucleic acids which are set free, e.g. by way of cell lysis and/or lysis of cellular organelles such as mitochondria, plastids, nuclei or other nucleic acid-containing organelles, can be purified by way of binding to a solid phase such as a mineral substrate, washing said mineral substrate with the bound nucleic acids and releasing said nucleic acids from said mineral substrate.
Adsorption of nucleic acids to glass particles or silica particles in the presence of chaotropic salts is known to the art (Vogelstein, B., and Gillespie, D., Proc. Natl. Acad. Sci. USA 76 (1979) 615-619) and provide the basis for chromatographic purification and separation processes for nucleic acids. Also known to the art are methods to isolate and purify RNA and DNA from lysates using high concentrations of chaotropic salts, e.g. sodium iodide, sodium perchlorate and guanidine thiocyanate (Boom, R., et al., J. Clin. Microbiol. 28 (1990) 495-503; Yamada, O., et al., J. Virol. Methods 27 (1990) 203-209). The purification of plasmid DNA from bacteria on glass dust in the presence of sodium perchlorate is described in Marko, M. A., et al., Anal. Biochem. 121 (1982) 382-387. In DE 37 24 442, the isolation of single-stranded M13 phage DNA on glass fiber filters by precipitating phage particles using acetic acid and lysis of the phage particles with perchlorate is described. The nucleic acids bound to the glass fiber filters are washed and then eluted with a methanol-containing Tris/EDTA buffer. A similar procedure for purifying DNA from lambda phages is described in Jakobi, R., et al., Anal. Biochem. 175 (1988) 196-201. The procedure entails the selective binding of nucleic acids to glass surfaces in chaotropic salt solutions and separating the nucleic acids from contaminants such as agarose, proteins or cell residue. To separate the glass particles from the contaminants, the particles may be either centrifuged or fluids are drawn through glass fiber filters. This is a limiting step, however, that prevents the procedure from being used to process large quantities of samples.
The use of magnetic particles to immobilize nucleic acids after precipitation by adding salt and ethanol is more advantageous and described e.g. in: Alderton, R. P., et al., Anal. Biochem. 201 (1992) 166-169 and WO 91/00212. In this procedure, the nucleic acids are agglutinated along with the magnetic particles. The agglutinate is separated from the original solvent by applying a magnetic field and performing a wash step. After one wash step, the nucleic acids are dissolved in a Tris buffer. This procedure has a disadvantage, however, in that the precipitation is not selective for nucleic acids. Rather, a variety of solid and dissolved substances are agglutinated as well. As a result, this procedure can not be used to remove significant quantities of any inhibitors of specific enzymatic reactions that may be present. Magnetic, porous glass is also available on the market that contains magnetic particles in a porous, particular glass matrix and is covered with a layer containing streptavidin. This product can be used to isolate biological materials, e.g., proteins or nucleic acids, if they are modified in a complex preparation step so that they bind covalently to biotin. Magnetizable particular adsorbents proved to be very efficient and suitable for automatic sample preparation. Ferrimagnetic and ferromagnetic as well as superparamagnetic pigments are used for this purpose. The most preferred MGPs are those described in WO 01/37291.
Purification of a nucleic acid by way of adsorbing the same to a substrate such as a mineral substrate in the presence of high concentration of salts is also applied to other complex mixtures. Examples therefor are known to the person skilled in the art of molecular biology and include reaction mixtures following, e.g., in-vitro synthesis of nucleic acids such as PCR, restriction enzyme digestions, ligation reactions, etc. In Vogelstein, B., and Gillespie, D., Proc. Natl. Acad. Sci. USA 76 (1979) 615-619, for instance, a procedure for binding nucleic acids from agarose gels in the presence of sodium iodide to ground flint glass is proposed. Another application for purification of a nucleic acid by way of adsorbing the same to a substrate such as a mineral substrate in the presence of a high concentration of salts is the removal of pyrogenic contaminants which may have copurified with the nucleic acid.
The mechanism by which nucleic acids bind to the mineral support in the presence of chaotropic agents is not entirely clear. It is hypothesized that the interaction between the nucleic acids and the solvent is influenced such that the nucleic acids adsorb to the mineral support and denaturate. In the presence of high concentrations of chaotropic agents the reaction is almost quantitative. The adsorbed nucleic acids can be eluted by applying to the mineral support buffers of low ionic strength.
U.S. Pat. No. 5,808,041 discloses methods for isolating nucleic acids with lengths greater than about 50 bases from certain biological samples. An aequous lysate containing chaotropic ions at a concentration above about 2 M is produced and the nucleic acids are adsorbed from the lysate to silica material (also referred to as “binding”). A slurry or resin comprising silica material and chaotropic salts is added to the biological material thus resulting in a one-step lysis and binding procedure. The methods for lysing a biological sample disclosed in the document include lysis of bacteria using alkali hydroxide and SDS, lysis of M13 or lambda phages by incubating the phages in the presence of 2.8 M guanidinium, and lysis of fresh or frozen tissue by incubating the tissue in the presence of 2.8 M guanidinium. Additionally, N-lauryl-sarcosine is used to aid lysing.
EP 0 389 063 and EP 0 819 696 disclose the method of purifying a nucleic acid by way of mixing in a liquid phase material containing the nucleic acid with a chaotropic substance and a nucleic acid binding solid phase. Thus, the procedures disclosed in the documents also represent one-step lysis and binding procedures. Following lysis and binding, the solid phase with bound nucleic acid is separated from the liquid phase. Following a washing step the nucleic acid is eluted from the solid phase. The documents disclose a lysis buffer containing about 10 M guanidinium thiocyanate, about 2% TRITON X-100 (Rohn & Haas Co.), about 0.1 M Tris salt, and about 50 μM EDTA. Another lysis buffer further contains 40% weight by volume dextrane sulfate. Another lysis buffer contains about 10 M guanidinium thiocyanate and about 50 μM EDTA. Other lysis buffers are disclosed in the documents that contain as a chaotropic substance potassium iodide or sodium iodide at a concentration of about 3 M, or potassium or sodium iodide in combination with 1 M or 8 M urea. Lysis of a biological sample is effected by way of incubating the sample with a lysis buffer, whereby 50 volume parts of the biological sample were mixed with 900 volume parts of a lysis buffer and 40 volume parts of a silica coarse. As an alternative to using silica coarse, other procedures are described wherein silica filter material is used.
EP 0 658 164 describes methods for the chromatographic purification of nucleic acids by way of chromatographic purification. Particularly, a two-step procedure comprising a first lysis step and a second binding step is described. In the first step (lysis), the biological sample is mixed with a chaotropic agent whereby the concentration of the chaotropic agente in the mixture is between about 2 M and about 4 M. Optionally, the mixture additionally contains phenol, chloroform or ether. Optionally, the mixture additionally contains a detergent. A protease is added and the mixture is incubated. In the second step (binding), an alcohol is added and the resulting mixture is contacted with the nucleic acid-binding solid phase.